Method for determining activity of bacterial nitrite reductase
A technology for the determination of nitrite and activity, which is applied in the field of biological enzyme detection, can solve the problems of expensive reagent operation process, cumbersome, laborious, etc., and achieve the effects of saving dosage, improving detection efficiency, saving manpower and testing time
Inactive Publication Date: 2013-08-14
SHANGHAI INST OF TECH
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Problems solved by technology
[0004] The purpose of the present invention is to provide a kind of nitrite reductase activity determination method in bacteria in order to solve the time-consuming, laborious, reagent-consuming and tedious technical problems of the operation process in the above-mentioned technology, namely to measure the biological sample by naphthalene ethylenediamine method commonly used The determination of medium nitrite is simplified, and the removal process of protein and lipid is improved. After the enzyme reaction, the general naphthalene ethylenediamine hydrochloride method is not used for colorimetric determination, but a multi-well plate and a multi-functional microplate reader are used for determination and analysis.
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Embodiment 1
[0066] Lactobacillus plantarum Lactobacillus plantarum A method for measuring the activity of nitrite reductase in medium, specifically comprising the following steps:
[0067] (1) Preparation of standard curve
[0068] ①. Preparation of reagents for standard curve preparation
[0069] Naphthaleneethylenediamine hydrochloride solution with a concentration of 0.2% (W / V):
[0070] Weigh 0.1g of naphthaleneethylenediamine hydrochloride, dissolve it in 50mL of distilled water, mix well, put it in a brown bottle, and store it in a refrigerator at 4°C in the dark, and prepare it immediately;
[0071] 0.4% (W / V) p-aminobenzenesulfonic acid solution:
[0072] Weigh 0.4g of p-aminobenzenesulfonic acid, dissolve in 100mL of 20% hydrochloric acid, mix well in a brown bottle, and store in the dark;
[0073] Sodium nitrite standard solution with a concentration of 0.2mg / mL:
[0074] Accurately weigh 0.1000g of sodium nitrite dried in a desiccator for 24 hours, add distilled water to ...
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Abstract
The invention discloses a method for determining the activity of a bacterial nitrite reductase. The method comprises the following steps: 1, making a standard curve; 2, extracting the bacterial nitrite reductase; 3, preparing a reaction mixed solution used in the determination process; 4, catalyzing a reaction by the bacterial nitrite reductase; and 5, determining the activity of the bacterial nitrite reductase: determining the light absorption value of a nitrite solution by a porous plate and an ELISA (enzyme linked immunosorbent assay) before and after the reaction, obtaining the NaNO2 content before and after the enzymatic reaction according to the standard curve, and calculating according to an enzyme vitality calculation formula to obtain the activity of the bacterial nitrite reductase to be determined. The method for determining the activity of the bacterial nitrite reductase of the invention, which adopts the porous plate and the ELISA after the enzymatic reaction, allows the analytic reagent dosage to be saved, manpower and test time to be simultaneously saved, and the detection efficiency to be substantially improved.
Description
technical field [0001] The invention belongs to the technical field of biological enzymatic detection, in particular to a method for measuring the activity of bacterial nitrite reductase. Background technique [0002] Nitrite reductase is an enzyme that reduces nitrite. Widely present in plants and microorganisms. Assimilative nitrite reductase contains siroheme, which performs 6-electron reduction to produce ammonia. The enzymes of higher plants, green algae and cyanobacteria use ferredoxin as electron donor. Spinach leaf nitrite reductase (molecular weight 60,000), containing siroheme, non-heme iron and acid-labile sulfur. Neurospora crassa nitrite reductase (molecular weight 290,000) and Escherichia coli nitrite reductase (molecular weight 190,000) contain FAD, non-heme iron and siroheme, with NAD(P)H as electron donor . Dissimilation-type enzymes participate in the process of nitrous acid oxidation of organic substances, in which the enzymes of denitrification bact...
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IPC IPC(8): G01N21/31
Inventor 龚钢明何婷婷高然
Owner SHANGHAI INST OF TECH